To achieve this goal, we constructed a modular metabolic pathway containing seven genes. In the engineered strain expressing this pathway, the percentage of odd-chain FAs out of total FAs was higher than in the control strain (3.86 vs. 0.84%). When this pathway was transferred into an obese strain, which had been engineered to accumulate large amounts of lipids, odd-chain fatty acid production was 7.2 times greater than in the control (0.05 vs. 0.36 g/L). This study shows that metabolic engineering research is making progress toward obtaining efficient cell factories that produce odd-chain FAs.Microbial oils are regarded as promising alternatives to fossil fuels as concerns over environmental issues and energy production systems continue to mount. Odd-chain fatty acids (FAs) are a type of valuable lipid with various applications: they can serve as biomarkers, intermediates in the production of flavor and fragrance compounds, fuels, and plasticizers. Microorganisms naturally produce FAs, but such FAs are primarily even-chain; only negligible amounts of odd-chain FAs are generated. As a result, studies using microorganisms to produce odd-chain FAs have had limited success. Here, our objective was to biosynthesize odd-chain FAs  To lessen the cost of producing microbial oils, considerable effort has been dedicated to enhancing production yield , tridecanoic acid (C13:0), and pentadecanoic acid (C15:0)—if grown with propionate-supplemented medium  from Salmonella enterica and acyl-ACP thioesterases (TEs) from Umbellularia californica and Ricinus communis. Additionally, propionate supplementation has allowed various yeasts  to produce odd-chain FAs   can be increased through the upregulation of threonine biosynthesis : from 0.006 to 0.246 g/L. This study further modified the experimental strain by replacing the native β-ketoacyl-ACP synthase (encoded by FabH) with one from Bacillus subtilis (encoded by FabHI) so that there was a biochemical preference for propionyl-CoA over acetyl-CoA  complex in the cytosol to see if it could improve the conversion of α-ketobutyrate to propionyl-CoA. This work demonstrates that our metabolic engineering strategy for directing metabolic fluxes through specific pathways can enhance odd-chain FA production.The objective of this study was to biosynthesize odd-chain FAs E. coli were previously described by Sambrook and Russell , and 2% (w/v) glucose. To complement strain auxotrophies, 0.1 g/L of uracil or leucine was added as necessary. To screen for hygromycin resistance, 250 μg/ml of hygromycin was added to the YPD. Solid media were prepared by adding 1.5% (w/v) agar.Media and growth conditions for  Russell ; those fillardin . Rich meillardin . The YNBWe used standard molecular genetic techniques  using the BamHI and AvrII restriction sites. The other four genes were cloned into two plasmids (JME4774 and JME4775) using Golden Gate assembly.For the cytosolic PDH complex, all the genes were synthesized and cloned in the plasmid pUC57 by GenScript Biotech . Cytosolic PHD1, the cassettes were constructed to include a promoter (pPHD1), a marker (URA3 or LEU2), and a terminator (TPHD1), which allowed the ORF gene to be removed via homologous recombination, as described in Papanikolaou et al. , and 6% (w/v) glucose. This medium had been placed in 250 mL Erlenmeyer flasks. The cultures were then incubated at 28°C and 180 rpm.The lipid biosynthesis experiments were carried out in minimal media, and the cultures were prepared as follows: an initial pre-culture was established by inoculating 10 mL of YPD medium in 50 mL Erlenmeyer flasks. Then, the pre-culture was incubated overnight at 28°C and 180 rpm. The resulting cell suspension was washed with sterile distilled water and used to inoculate 50 mL of minimal medium YNBD6 containing 0.17% (w/v) yeast nitrogen base , 0.15% (w/v) NHLipids were extracted from 10 to 20 mg of freeze-dried cells and converted into FA methyl esters (FAMEs) using the procedure described by Browse et al. . The FAMTo determine dry cell weight (DCW), 2 mL of the culture was taken from the flasks, washed, and lysophilized in a pre-weighed tube. The differences in mass corresponded to the mg of cells found in 2 mL of culture.Y. lipolytica. As shown in E. coli. Lee et al. showed that levels of odd-chain FAs could be increased by introducing the threonine biosynthesis pathway , especially when mutated homoserine dehydrogenase  was also expressed .Propionyl-CoA is a key primer in the synthesis of odd-chain FAs. It can be synthesized using β-oxidation from direct precursors, propionate, or long-chain FAs. It can also be created from other metabolites via several metabolic pathways, such as the citramalate/2-ketobutyrate pathway, the aspartate/2-ketobutyrate pathway, the methylmalonyl-CoA pathway, the 3-hydroxypropionate pathway, and the isoleucine or valine degradation pathway . Here, wAAT2; the homoserine synthesis module (module H), which included HOM3, HOM2, and HOM6; and the threonine synthesis module (module T), which included THR1, THR4, and ILV1. While threonine and α-ketobutyrate (module T) are synthesized in the mitochondria in S. cerevisiae, the same may not be true in Y. lipolytica. While the locations of the relevant enzymes are as yet unknown in Y. lipolytica, predictive analyses  in the pathway was overexpressed in Y. lipolytica both individually and in tandem. The strain with the full modular pathway (ATH) was constructed by removing and reusing the URA3 marker —from oxaloacetate to α-ketobutyrate—by overexpressing seven genes . There wanalyses  suggest assembly . They we3 marker . We veri3 marker .AAT2, and the WT-T strain, which overexpressed THR1, THR4, and ILV1, total lipid content  was lower than in the wild-type (WT) strain; the percentage of odd-chain FAs out of total FAs was similar  than that in the WT strain (0.84%) , which have been found to positively influence lipid synthesis  . For theY. lipolytica strain that received propionate supplementation  genes . These results suggest that disrupting the methylcitrate cycle does not provide the benefits seen previously , E2 , and E3 . There is a fourth component  that binds to and positions E3 relative to E2. In Y. lipolytica, the PDH complex is found in mitochondria and catalyzes the pathway from pyruvate to acetyl-CoA. A few studies have examined the functional expression of the PDH complex in Y. lipolytica. One study attempted to overexpress the direct pathway from pyruvate to acetyl-CoA in a coordinated manner  improved α-ketoglutarate production  via Golden Gate assembly resulted in higher levels of odd-chain FAs being produced from glucose. The best strain generated a level of odd-chain FAs, 0.36 g/L in flask that is the highest to date to be achieved in Nielsen, .Y. lipolytica to improve odd-chain FA production could be useful in a wide range of pharmaceutical and industrial contexts.Before the approach described here can be applied at industrial scales, certain issues must be resolved. It is necessary to perform research in which combinatorial pathway analysis (Lütke-Eversloh and Stephanopoulos, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.YP, RL-A, and J-MN planned the study. YP designed and carried out the experiments and drafted the manuscript. All the authors revised and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


V=const) on the MSC-Scaffold prototype spikes to achieve a biomineral coating with a native Ca/P ratio. The CaP ECDV=const process on the MSC-Scaffold Ti4Al6V pre-prototypes was investigated for potential VECDfrom −9 to −3 V, and followed by 48 h immersion in a simulated body fluid. An acid–alkaline pretreatment (AAT) was applied for a portion of the pre-prototypes. Scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and X-ray diffraction (XRD) studies of deposited coatings together with coatings weight measurements were performed. Themost suitable VECD range, from −5.25 to −4.75 V, was determined as the native biomineral Ca/P ratio of coatings was achieved. AAT increases the weight of deposited coatings  and the coverage degree of the lateral spike surfaces . XRD confirmed that the multiphasic CaP coating containing crystalline octacalcium phosphate is produced on the lateral surface of the spikes of the MSC-Scaffold. ECDV=const preceded by AAT prevents micro-cracks on the bone-contacting surfaces of the MSC-Scaffold prototype, increases its spikes’ lateral surface coverage, and results in the best modification effect at VECD = −5.00 V. To conclude, the biomimetic MSC-Scaffold prototype with desired biomineral coating of native Ca/P ratio was obtained for cementless RA endoprostheses.The multi-spiked connecting scaffold (MSC-Scaffold) prototype with spikes mimicking the interdigitations of articular subchondral bone is an essential innovation in surgically initiated fixation of resurfacing arthroplasty (RA) endoprosthesis components. This paper aimed to present a determination of the suitable range of conditions for the calcium phosphate (CaP) potentiostatic electrochemical deposition (ECD Resurfacing joint endoprostheses, e.g., total resurfacing hip arthroplasty (THRA) endoprostheses, are a bone-tissue-preserving option offered for relatively young and active patients with advanced osteoarthritis (OA). In contrast to traditional long-stem total hip replacement (THR) endoprostheses requiring surgical removal of the femoral head and neck, for the current generation of THRA endoprostheses, the femoral head is not removed, but is instead trimmed and capped with metal components fixed in subchondral bone with cement and a short stem placed in the femoral neck ,2. An esUnmodified metallic surfaces of joint endoprostheses components interacting with the bone have low osteoconductive and osseointegrative behavior; therefore, surface modifications are essential to enhance their biocompatibility and biological performance. Calcium phosphate (CaP) bioceramics are widely used in the field of bone regeneration, both in orthopaedics and in dentistry, due to their good biocompatibility, osteoconductivity, and osseointegrativity ,15,16,17Synthetic CaP coatings can be prepared using a variety of processes. In general, the commonly used methods can be divided into two groups, physical deposition techniques and wet-chemical techniques ,18. PhysCaP coating deposition on flat substrates has been widely investigated, while CaP deposition on the bone-contacting surfaces of complex geometrical shapes, e.g., of porous implants or additively manufactured scaffolds, have only been studied relatively rarely and quite recently ,40,41. MThe ECD process can be carried out at room temperature and allows for the CaP surface modification of complex-shaped Ti-alloy implants, resulting in a non-delaminating CaP coating of ca 1 μm thickness characterized by relatively high adhesive strength in comparison with the EPD process, where the hydroxyapatite (HA) coating is obtained from a suspension containing HA particles. Without applying thermal post-processing by subsequent sintering, the EPD deposited HA coating delaminates ,24,30,313)2, and ammonium dihydrogen phosphate, NH4H2PO4, wherein the weight ratio of calcium to phosphorus is ca. 1.67 and is the same as the ratio of Ca/P in the native osseous CaPs [In the ECD process, CaP coatings are formed from an electrolyte containing calcium nitrate, Ca showed significantly higher repeatability than the galvanostatic ECD process [V=const process on the MSC-Scaffold pre-prototypes.Attempts to modify the bone-contacting surfaces of the MSC-Scaffold pre-prototype by ECD of CaPs has been undertaken initially at constant current densities with subsequent immersion in SBF  . It was  process . SimilarThe particular aim of this paper was to present the determination of the suitable range of conditions for potentiostatic electrochemical deposition of calcium phosphates (CaPs) on the MSC-Scaffold prototypes to achieve a native biomineral Ca/P ratio in the coating, which is of great importance for good biocompatibility and biological performance of the implant in vivo. Good biological performance of this implant was proved in our recent investigation in swines on a partial knee arthroplasty (RKA) endoprosthesis working prototype with the CaP coated in potentiostatic ECD process MSC-Scaffold .The main aim of our work is to elaborate on the suitable MSC-Scaffold prototypes for a new generation of entirely cementless RA endoprostheses.Sc200) and 350 μm (PSc350), both circumferentially and radially, which corresponds to the thickness of bone trabeculae of cancellous bone. In 3.Surface modification of the MSC-Scaffold prototype for non-cemented resurfacing joint endoprostheses was carried out on the MSC-Scaffold pre-prototypes designed as fragments of the central part of the femoral component of the TRHA endoprosthesis. The multilateral spikes of the MSC-Scaffold pre-prototypes were arranged in concentric parallel rings around the central spike with axes parallel to each other, whereas the central spike was coincident with the femoral head axis of symmetry. The length of the square side in the spike pyramid’s base was 0.5 mm in the MSC-Scaffold CAD model. The prototype THRA endoprosthesis with the MSC-Scaffold manufactured using selective laser melting (SLM) of Ti4Al6V powder is presented in After SLM manufacturing, to remove the adhered powder aggregates from the spike surfaces, a manual blasting treatment was carried out using an experimentally customized abrasive mixture composed of equal proportions of white aloxiteF220 (~53–75 μm), white aloxite F320 (~29.2 μm ± 1.5%), and blasting micro glass beads (~30 μm ± 10%) . CleaninECD) for the ECDV=const process, a total of 56 MSC-Scaffold pre-prototypes (28 of each variant) were subjected to surface modification. To search for the appropriate conditions of the ECDV=const process, VECD values from −9 to −3V were investigated using a potentiostat-galvanostat apparatus . The CaP ions were deposited from a solution composed of 0.042 M calcium nitrate, Ca(NO3)2, and 0.025 M ammonium dihydrogen phosphate, NH4H2PO4, with pH=6. The ECD process was performed in a two-electrode system. The process was carried out for one hour at room temperature. A gold plate anode was used as the counter electrode. After the ECDV=const process, the MSC-Scaffold pre-prototypes, playing the role of working electrode, were rinsed with distilled water and, to convert the deposited amorphous CaP coating into the bone-like biomineral coating, they were immersed for 48 h in an SBF solution composed of 6.8 g/L NaCl, 0.4 g/L KCl, 0.2 g/L CaCl2, 0.2048 g/L MgSO4∙7H2O, 0.1438 g/L NaH2PO4∙H2O and 1.0 g/L NaHCO3 at 37 °C. After the incubation in SBF, the MSC-Scaffold pre-prototypes were dried at room temperature and their final weight was measured. The weight increase due to surface coatings deposited on the spikes of the MSC-Scaffold pre-prototypes was calculated as the difference between the initial and final weights of the modified MSC-Scaffold pre-prototype. An analysis of chemical composition of the coating deposited on the lateral spike surfaces of the MSC-Scaffold pre-prototypes was performed using a scanning electron microscope  equipped with the energy dispersive X-Ray (EDS) system . The results can be used for calculating the Ca/P ratios.To determine the most suitable range of potential . Structure and phase composition of the deposited coating was identified by XRD on a PANalytical EMPYREAN X-ray diffractometer  at a scanning speed of 0.02°/s with Cu Kα radiation  at a 2θ range of 30–70°. Since, there was no technical possibility to analyze the surface of MSC-Scaffold pre-prototypes directly, so to obtain the XRD roentgenograms we had to use the deposits detached mechanically from the spikes as a powder sample.Sc200 and the PSc350MSC-Scaffold pre-prototypes as a function of the applied VECD values during the ECDV=const process of CaP deposition.Sc200MSC-Scaffold pre-prototypes modified using VECD values ranging from −9 to −5.25 V, the surface weight increase was about 3 mg for the initial stages (from 2.75 mg for VECD = −9 V to 3.65 mg for VECD = −7 V). Increasing the VECD value past −5.25 V resulted in reducing the deposited coating weight increase to 2 mg (for VECD = −4.75 V), while for VECD = −4.5 V and VECD = −3 V there was no noted weight increase. EDS analysis of chemical composition confirmed the absence of Ca and P on the lateral spike surfaces of the MSC-Scaffold pre-prototypes modified by applying VECD values of −4.5 V and −3 V. SEM analysis revealed that, for the PSc200 MSC-Scaffold pre-prototypes for which a weight increase was observed, CaPs were deposited only on the upper regions of spikes. In this case, a significant amount of CaP deposit was found in the inter-spike space of the MSC-Scaffold pre-prototypes. This phenomenon was judged to be disadvantageous. Example SEM photographs showing this effect are presented in Sc200 MSC-Scaffold pre-prototypes corresponds to the Ca/P ratio characteristic for CaPs. EDS analysis shows that the Ca/P ratios reached values below 1.00 and 3.73, so in this case, there was no CaP coating on the spike surfaces, but only Ca and P ions randomly deposited onto the surface of the MSC-Scaffold pre-prototypes’ spikes. In the first case, almost the entire surface was deposited with Ca whereas in the second case, nearly all deposits were P. At −4.5 V no mass increase was observed. For the PV=const surface modification of the PSc200 MSC-Scaffold pre-prototypes led to the decision to abandon further research using this geometrical variant of MSC-Scaffold pre-prototype.An example of the EDS chemical mapping of two magnified areas is presented in Sc350 MSC-Scaffold pre-prototypes modified using VECD values ranging between −9 to −5.50 V, the weight increase of the surface coating was low (less than 1 mg) and the Ca/P ratios determined in the deposited surface coatings did not correspond to the Ca/P values characteristic for CaPs. A significant increase in weight (about 5 mg) was found when applying VECDvalues ranging between −5.25 to −4.75V. For the ECD process carried out using VECD values above −4.50 V, a slight weight increase was observed (approximately 0.50–0.75 mg). Unfortunately, the Ca/P ratio in the deposited surface coating did not correspond to the characteristic Ca/P values for native osseous CaPs. EDS analysis of all the PSc350 MSC-Scaffold pre-prototypes modified using VECD values of −5.25, −5.00 and −4.75 V confirmed the presence of CaPs having the Ca/P ratios consistent with the native osseous CaPs). Therefore, VECD values from −5.25 to −4.75V can be recommended as the most suitable conditions for the CaP ECDV=constsurface modification of the PSc350 MSC-Scaffold pre-prototypes.For the PSc350 MSC-Scaffold pre-prototypes modified using the VECD values of −5.25, −5.00 and −4.75 V. The dependence was determined both for the MSC-Scaffold pre-prototypes that underwent the AAT pretreatment and those that did not.Sc350 MSC-Scaffold pre-prototypes was obtained for VECD = −5.00 V. It can be clearly seen from ECD = −5.25 V, by 9% for VECD = −5.00 V and by 15% for VECD = −4.75 V). In both cases, the highest average weight increase for the modified PSc350SEM images of the lateral spike surfaces of the MSC-Scaffold pre-prototypes modified by a one hour ECDV=const process carried out using VECD values of −5.25, −5.00, and −4.75 V, followed by 48 h incubation in SBF, without AAT pretreatment (reatment a–c and wreatment d–f.V=const process carried out without AAT pretreatment is non-uniform and seems to be unstable (the surface is not consistent). For MSC-Scaffold pre-prototypes modified using −5.25 V, most of the lateral spike surfaces remained uncoated in their medial part. The coating was deposited mostly on the upper part of the spikes. For the remaining pre-prototypes  the entire lateral surface of spikes was CaP coated, but numerous micro-cracks, especially for VECD = −5.00 V, were noted. SEM analysis of the microstructure of the lateral spike surfaces shows that the CaP coating obtained during the ECDECD values of the ECDV=const process. Plate-like and needle-like shaped CaP crystals appear on the lateral surfaces of the MSC-Scaffold pre-prototypes. In particular, a significant accumulation of such crystals can be observed in the upper part of the MSC-Scaffold’s spikes. As can be clearly seen in the SEM images presented in Sc350 MSC-Scaffold pre-prototypes that underwent one hour of ECDV=const process carried out using VECD values of −5.25, −5.00 and −4.75 V followed by 48 h immersion SBF, with and without AAT pretreatment as a function of the applied VECD during the ECDV=const process. From the EDS analysis, the molar ratios of calcium to phosphorous on the lateral spike surfaces were 1.58–1.74, which is consistent with the values of native osseous CaP. The graph in Sc350 MSC-Scaffold pre-prototypes modified at VECD = −5.00 V (average 68±6%). For other VECD values, the coverage degree of the lateral spike surfaces was half the size (33±5–35±5%). Applying of AAT pretreatment increases the coverage degree of the lateral spike surfaces .The EDS mapping results for the modified lateral spike surfaces and the quantitative analysis performed using ImageJ show that the greatest lateral spike surface coverage degree was obtained for the PECD = −5.00 V, an XRD analysis was performed. The results are shown in To confirm the crystalline form of CaP was deposited on the surface of MSC-Scaffold pre-prototypes due to electrochemical treatment using V8H2(PO4)6∙5H2O (PDF2# 00-026-1056), calcium metaphosphate, Ca(PO3)2 (PDF2# 00-003-0348), and monocalcium phosphate monohydrate, Ca(H2PO4)2∙H2O (PDF2# 00-003-0284). Apart from this, the impurity phases from the SBF solution were found on the surface sodium chloride, NaCl (PDF2# 01-077-2064), and sodium hydrogen carbonate, NaHCO3 (PDF2# 00-021-119). The results of the XRD investigation showed that, during the combined ECD process conducted under the determined conditions, CaP biomineral coating is produced on the lateral surface of spikes of the MSC-Scaffold.As one can see, the obtained CaP coating is multiphasic and there are identified peaks attributed to CaP phases like octacalcium phosphate, CaV=constprocess. Since the MSC-Scaffold prototype, as the original concept of a multi-spiked  fixation of RA endoprostheses components, was developed within the frames of two of our research projects, its most suitable geometrical properties evolved based on the findings of bioengineering research in relation to that primarily suggested in the patented version [V=const) showed much higher repeatability compared to those of the galvanostatic process (ECDj=const).In this study, the experimental CaP modification of the bone-contacting surfaces of MSC-Scaffold pre-prototypes was undertaken in search of the suitable range of conditions for CaP deposition in the ECD version ,4,5. Add version , i.e., i version .Signific version . After tV=const process. VECD values from the −9 to −3V range were applied. This stage of investigations showed that the suitable conditions for the ECDV=const process of CaP modification of complex-shaped bone-contacting surfaces of the MSC-Scaffold prototype are strongly influenced by the geometrical features of the scaffold prototype, i.e., by the distance between the spikes. In the case of insufficient room between the MSC-Scaffold’s spikes, the CaP deposits are found between the spikes instead of on their lateral surface. Hence, the PSc200variant of the MSC-Scaffold pre-prototype was excluded from further research. Based on the characterization of the coating’s physiochemical properties—in terms of structural (EDS) and morphological (SEM) properties, and the weight increase of the deposits—the VECD range from −5.25 to −4.75V was determined to provide the expected CaP modification of the bone-contacting surfaces of the PSc350 variant of the MSC-Scaffold pre-prototype.Experimental CaP modification of the MSC-Scaffold pre-prototypes was carried out in two steps. In the first step, the purpose was to determine the most suitable range of conditions for the ECDECD values that achieve the native biomineral Ca/P ratio in coatings on the lateral surface of the MSC-Scaffold pre-prototypes. The investigation procedure applied in the first step was extended to include EDS surface mapping and quantitative analysis of crystalline phases (XRD). The enhancement of the coverage degree of the lateral spike surfaces and the coverage uniformity were ascertained. AAT pretreatment prevents micro-crack formation on the bone-contacting surfaces of the MSC-Scaffold and also affects the increase of the spikes’ lateral surface coverage. The Ca/P ratios of deposits on the lateral spike surfaces in all modified MSC-Scaffold pre-prototypes are consistent with the Ca/P ratios of native osseous CaPs, and plate-like and needle-like CaP crystals appeared on the bone-contacting surface of the MSC-Scaffold pre-prototypes undergoing the AAT pretreatment.In the second step, the influence of AAT pretreatment was examined by applying the previously determined range of VECD = −5.00 V—the native biomineral Ca/P ratio of deposits  was achieved, as well as the highest average mass growth of the coating and the highest coverage degree of spikes’ lateral surface (even in the case of the MSC-Scaffold prototypes without AAT pretreatment). The numerous micro-cracks observed on the MSC-Scaffold pre-prototypes CaP modified at VECD= −5.00V were prevented by applying the AAT pretreatment, finally providing the highest uniformity in comparison to the other VECD values.The best overall results for CaP modification of the bone-contacting surfaces of the MSC-Scaffold pre-prototypes were obtained for the VSc200) and 350 μm (PSc350), the PSc200 MSC-Scaffold variant appeared to be inappropriate to be CaP modified in the ECDV=const process.The effect of CaP ECD deposition on the MSC-Scaffold prototype is conditioned not only by the appropriate choice of electric parameter values of the ECD process but also on the geometrical features (distance between the spikes) of the MSC-Scaffold. Of the two examined design variants, the distance between spike bases of 200 μm (PSc350MSC-Scaffold pre-prototypes, the most suitable VECD values for the ECDV=constprocess were from −5.25 to −4.75 V and the best results for CaP modification of their bone-contacting surfaces was obtained at VECD = −5.00 V; the native biomineral Ca/P ratio of coatings was achieved for all the VECD values used.Based on the SEM and EDS studies of deposited CaP coatings together with the measurements of the weight increase for the PECD = −5.00 V.ECD combined with AAT pretreatment prevents micro-crack formation on the bone-contacting surfaces of the MSC-Scaffold prototype and increases the spikes’ lateral surface coverage; the best results for the CaP modification of the bone-contacting surfaces was obtained at VThus, the biomimetic MSC-Scaffold prototype with desired biomineral coating of native Ca/P ratio on bone-contacting surfaces was obtained for a new kind of entirely non-cemented resurfacing arthroplasty endoprostheses.


End-stage renal disease (ESRD) strongly associates with cardiovascular disease (CVD) risk. This risk is not completely mitigated by renal replacement therapy. Endothelial dysfunction (ED) and low-grade inflammation (LGI) may contribute to the increased CVD risk. However, data on serum biomarkers of ED and LGI during the transition to renal replacement therapy  are scarce.We compared serum biomarkers of ED and LGI between 36 controls, 43 participants with chronic kidney disease (CKD) stage 5 non-dialysis (CKD5-ND), 20 participants with CKD stage 5 hemodialysis (CKD5-HD) and 14 participants with CKD stage 5 peritoneal dialysis (CKD5-PD). Further, in 34 and 15 participants repeated measurements were available during the first six months following dialysis initiation and kidney transplantation, respectively. Serum biomarkers of ED  and LGI  were measured with a single- or multiplex array detection system based on electro-chemiluminescence technology.In linear regression analyses adjusted for potential confounders, participants with ESRD had higher levels of most serum biomarkers of ED and LGI than controls. In addition, in CKD5-HD levels of serum biomarkers of ED and LGI were largely similar to those in CKD5-ND. In contrast, in CKD5-PD levels of biomarkers of ED were higher than in CKD5-ND and CKD5-HD. Similarly, in linear mixed model analyses sVCAM-1, thrombomodulin, sICAM-1 and sICAM-3 increased after PD initiation. In contrast, incident HD patients showed an increase in sVCAM-1, P-selectin and TNF-α, but a decline of hs-CRP, SAA and IL-6. Further, following kidney transplantation sVCAM-1, thrombomodulin, sICAM-3 and TNF-α were lower at three months post-transplantation and remained stable in the three months thereafter.per se. In contrast to dialysis, several serum biomarkers of ED and LGI markedly declined following kidney transplantation.Levels of serum biomarkers of ED and LGI were higher in ESRD as compared with controls. In addition, PD initiation and, less convincingly, HD initiation may increase levels of selected serum biomarkers of ED and LGI on top of uremia  Individuals with end-stage renal disease (ESRD) are at high risk of cardiovascular disease (CVD) mortality,2. RenalEndothelial dysfunction (ED) and low-grade inflammation (LGI) have both been implicated in the adverse CVD risk associated with ESRD–10. Indei.e., including assessment of ED and LGI prior and following exposure to dialysis)Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: PartlyReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: I Don't KnowReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: NoReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuscript is of interest providing some interesting data on the evolution of biomarkers of endothelial function and inflammation, but many concerns are present.General comments: The manuscript is very difficult to read with a tendency to be confused. Statistical analysis must be reviewed by a specialist.The abstract must be reviewed. The methods and results are not clear and the conclusion I is not really conclusive. Word  you have 3 with monotherapy with tacrolimus, 5 with tacro and MMF and 7 with Tacro MMF and corticoids. It is not clear for me for patients with everolimus if they are still with everolimus or not.Pour les dilyser penser à discuter les différentes membranes.In results,In table 1, the authors must separate HD and PD patients. In controls and CKD5-ND it is not residual urine outpout but diuresis. In this table, in the 18% of patient with CKD have a history of Ktx, did they have an immunosuppressive drugs? This must be stated and clear in the characteristics of the patients. It is very different to study endothelial dysfunction to have or not IS drugs.Important clinical data are missing: tobacco, alcool, lipids concentration, calcium phosphore bicarbonate potassium, hb etc. Medications used, mainly immunosuppressive drugs and corticoids, antihypertensive drugs, HMG CoA inhibitors, anticoagulants, aspirin etc. All this drugs could play an important role on endothelial function and inflammation.For the table 2, supplementary analysis separating HD and PD must be done. Adjustment with blood pressure could be of interest mainly regarding endothelial dysfunction.For the longitudinal analysis we must have separation of PD and HD patients. In addition, in my opinion for dialysis patient regarding the boxplot there is no real differences in all the biomarkers, the test used for statistical significance must be provide explicitly in the results. More interesting than a box blot the authors must provide individual courses of the biomarkers separating HD and PD.We must have comparisons with other biological values, like calcium phosphore, bicarbonate urea, creatininemia etc.For transplantation patients same remarks regarding the representation of data. It could be of interest to indicate the course of values for the 4 patients undergoing dialysis then transplantation the data.The additional analysis section is a non sense. All this analysis must be included with main analysis or not discusses. The patients with cyst infection must be definitively excluded.The discussion will be analyzed after the modification asked for the methods and results.Reviewer #2: In the present study, the authors compared biomarkers of endothelium dysfunction and low-grade inflammation in controls and in different groups of CKD patients. They show that most biomarkers are higher in CKD patients than in controls, higher in dialysis patients than in non-dialyzed patients and decline after kidney transplantation. Higher levels of serum biomarkers of ED and LGI in CKD patients have been previously shown in the literature. In addition, the data on serum biomarkers after kidney transplantation do not provide new informations and only confirm previous studies.1) The design of the study is complex. It mixes the data of three separate observational studies, but the number of subjects per group is quite low. The characteristics of CKD5-ND and CKD5-D patients are different in terms of origin of end stage renal disease, and presence of diabetes mellitus.2) Patients on PD are different from patients on HD , and additional analysis demonstrated differences in biomarkers in these two groups of patients. Therefore,the authors should not include these patients in the same CKD5D group.3) The choice of statistical analyses is not clear. Why do the authors compare the data of biomarkers between groups with linear regression analysis and not with tests for group comparison like ANOVA or Mann Whitney? In addition, why do the authors use a natural log transformation to perform statistical test on the levels of serum biomarkers? How is the distribution of the data ?4) Identical data are presented several times, in different figures/tables. For example, Figure 2 and Table 3 provides the same informations, data in Table 3 are also in S1 Table etc…5) For a better understanding, the authors should add the symbols of p values on figures and tables. In particular, the p values between CKD5-ND and CKD5D should be mentioned in Table 1.6) In table 3, an entire column is NA, please remove the column. In S1 Table, dialysis vintage and KT/V are NA, please remove the lines.Reviewer #3: Manuscript is well written. Only minor suggestions to improve the manuscript.Please try to avoid the use of  in the descriptive sentences in academic writing “This risk is not (completely) mitigated by renal replacement therapy”“Only data of their first study was analyzed” “of” should be “from”Also, I suggest the investigators to may improve the introduction/discussion by going over very nice review on this topic PMID: 30607032 Nat Rev Nephrol. 2019 Feb;15(2):87-108. doi: 10.1038/s41581-018-0098-z**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  8 Aug 2019van Gennip et al., PLOS ONEManuscript ID: PONE-D-19-18004August 8, 2019Dear editor, dear reviewers,Thank you for providing us an opportunity to revise our manuscript. We thank the editor and the reviewers for their thoughtful comments and suggestions which have helped us to improve our manuscript. For your convenience, please find below, in bold, our point-by-point rebuttal. All changes made in the manuscript are highlighted with ‘tracked changes’. The page numbers of the manuscript are used below to indicate where the changes have been made. On behalf of all authors,Yours sincerely,April van Gennip, MD Remy Martens, MD, PhD Response to the Reviewer #1The manuscript is of interest providing some interesting data on the evolution of biomarkers of endothelial function and inflammation, but many concerns are present.General comments: The manuscript is very difficult to read with a tendency to be confused. Statistical analysis must be reviewed by a specialist. We thank the reviewer for the time spend to review the manuscript.1) The abstract must be reviewed. The methods and results are not clear and the conclusion I is not really conclusive. We have reviewed and updated the abstracts within the constraints of the word limits :“Introduction: End-stage renal disease (ESRD) strongly associates with cardiovascular disease (CVD) risk. This risk is not completely mitigated by renal replacement therapy. Endothelial dysfunction (ED) and low-grade inflammation (LGI) may contribute to the increased CVD risk. However, data on serum biomarkers of ED and LGI during the transition to renal replacement therapy  are scarce. Methods: We compared serum biomarkers of ED and LGI between 36 controls, 43 participants with chronic kidney disease (CKD) stage 5 non-dialysis (CKD5-ND), 20 participants with CKD stage 5 hemodialysis (CKD5-HD) and 14 participants with CKD stage 5 peritoneal dialysis (CKD5-PD). Further, in 34 and 15 participants repeated measurements were available during the first six months following dialysis initiation and kidney transplantation, respectively. Serum biomarkers of ED  and LGI  were measured with a single- or multiplex array detection system based on electro-chemiluminescence technology.Results: In linear regression analyses adjusted for potential confounders, participants with ESRD had higher levels of most serum biomarkers of ED and LGI than controls. In addition, in CKD5-HD levels of serum biomarkers of ED and LGI were largely similar to those in CKD5-ND. In contrast, in CKD5-PD levels of biomarkers of ED were higher than in CKD5-ND and CKD5-HD. Similarly, in linear mixed model analyses sVCAM-1, thrombomodulin, sICAM-1 and sICAM-3 increased after PD initiation. In contrast, incident HD patients showed an increase in sVCAM-1, P-selectin and TNF-α, but a decline of hs-CRP, SAA and IL-6. Further, following kidney transplantation sVCAM-1, thrombomodulin, sICAM-3 and TNF-α were lower at three months post-transplantation and remained stable in the three months thereafter.Conclusions: Levels of serum biomarkers of ED and LGI were higher in ESRD as compared with controls. In addition, PD initiation and, less convincingly, HD initiation may increase levels of selected serum biomarkers of ED and LGI on top of uremia per se. In contrast to dialysis, several serum biomarkers of ED and LGI markedly declined following kidney transplantation.”2) Word : “This risk is not (completely) mitigated by renal replacement therapy.”3) Ref 3 is of no interest and must be removed.We agree. This reference was inserted erroneously and we have now removed this reference.4) In methods part, the timing of blood samples collection must be more precise. First the vintage of collection must be given for all the patients. It is not entirely clear what the reviewer means with ‘vintage of collection’. All samples were collected during the period the study was conducted. This is described in the methods section in the paragraph study population and design. The studies included were conducted between February 2012 and July 2017, between October 2013 and January 2018, and between June 2012 and December 2017, respectively. 5) Second, the timing of collection regarding the dialysis séance must be precise, first, second or third dialysis of the week.In the group of prevalent dialysis patients samples were collected prior to each patients’ mid-week dialysis session. In the group of incident dialysis patients the timing of blood sampling relative to the dialysis session was not standardized . The same held true for kidney transplant patients who were on dialysis prior to KTx. We have added this information to the Methods section : “In participants on HD, study measurements and blood sampling were performed prior to one of their dialysis sessions .”6) Third, for the biomarkers, how the sample were conserved? All the measurements were done at the same time? Did the authors have data on the effects of blood conservation on the concentration of the biomarkers?Blood was collected in sterile 5 ml BD vacutainer SST II Advance tubes. After centrifugation, serum was aspirated and stored at -80°C until analysis. Before analysis, serum samples where thawed and mixed thoroughly. We have added this information to the Methods :” Blood was collected in sterile 5 ml BD vacutainer SST II Advance tubes. After centrifugation, serum was aspirated and stored at -80°C until analysis. Before analysis, serum samples where thawed and mixed thoroughly.”All measurements were done at the same time. Biomarkers were analyzed with 6 multiplex assays. Per assay, analyses were performed within the same batch to avoid batch effects. In addition, combining the analyses of two assays reduced the number of freeze-thaw cycles to three in total. Unfortunately, data on long-term stability of the serum biomarkers are lacking, except for hs-CRP, which is stable up to 11 years if stored at -80°C [1].7) For others biological value the technique of dosage must be provide. It is not clear to us what the reviewer means with ‘technique of dosage’. 8) Finally, the kidney transplantation protocol is very different with three patient included in a randomized trial. I think these patients must be excluded.All patients received standard protocol patient care during the kidney transplant period. The only difference is the use of different immunosuppressive protocols. However, the immunosuppressive medication as described in the manuscript and used in the RCT (TRANSFORM) and the observational study are already part of standard patient care. Therefore, we chose to include these patients into the analyses since our study has an observational nature and as such therapy cannot be fully standardized. 9) The values, especially for inflammation markers are very difficult to interpret when one a small sample of patients (15) you have 3 with monotherapy with tacrolimus, 5 with tacro and MMF and 7 with Tacro MMF and corticoids. It is not clear for me for patients with everolimus if they are still with everolimus or not.The immunosuppressive regimen, albeit protocolized, is tailored towards the individual patient’s clinical response, leading to some variation in immunosuppressive medication. This is inherent to clinical practice and the observational nature of this study.However, we have mentioned this as a study limitation in the Discussion : “Fifth, in this observational study, the immunosuppressive regimen was protocolized but not fully standardized, which may hamper the interpretation of results in kidney transplant recipients.Two patients started with everolimus. One patient was lossed-to-follow-up, the other patient used everolimus throughout the entire study period. Which is now stated more clearly in the S1 methods: “Three patients included in this study were simultaneously enrolled in a different study (TRANSFORM) (NCT01950819). Two of these three patients were randomized to receive tacrolimus, everolimus and prednisolone as triple therapy from time of kidney transplant. One of these two patients withdrew informed consent for further follow-up in our study in the first week after kidney transplantation for personal reasons. The other received everolimus throughout the entire study period. The third patient was randomized to receive tacrolimus (prograft), MMF and prednisolone as triple therapy from time of kidney transplant. This patient switched to the local immunosuppressive protocol of our centre after the 6 month follow up visit.”10) Pour les dilyser penser à discuter les différentes membranes. It is not entirely clear what the reviewer means with this. An overview of the dialysis membranes used for the patients in this study is provided in S1 Methods .11) In results, In table 1, the authors must separate HD and PD patients. In controls and CKD5-ND it is not residual urine outpout but diuresis. Thank you for noticing. We have changed ‘residual urine output’ into ‘diuresis/ residual urine output’ in Table 1 and S1 Table. In addition, we now present characteristics in Table 1 stratified according to controls, CKD5-ND, CKD5-HD and CKD5-PD. Similarly, we now present characteristics in S2 Table stratified according to incident HD, incident PD and kidney transplant recipients.The description of the cross-sectional population characteristics in the Results  has been adapted as well: “Thirty-six controls, 43 participants with CKD5-ND, 20 participants with CKD5-HD and 14 participants with CKD5-PD comprised the cross-sectional study population . In general, participants with CKD5-ND, CKD5-HD and CKD5-PD were more often men and had a worse CVD risk profile than controls.Participants with CKD5-HD and CKD5-D more often had diabetes mellitus than those with CKD5-ND. All participants with CKD5-ND and most with CKD5-D had residual urine output. Participants with CKD5-PD slightly younger, less often men, less often had a history of CVD, and had higher residual GFR , slightly more fluid overload and shorter dialysis vintage than participants with CKD5-HD, although not all differences were statistically significant in this relatively small study population.”Similarly for the description of the longitudinal population characteristics : “As compared with kidney transplant recipients, incident HD and PD patients were older (65.1 ±12.0 and 57.1 ±12.1 years vs. 51.6 ±12.8 years) and more often had a history of CVD (38.9% and 31.2% vs. 13.3%). In addition, incident HD patients were more often men (83.3% vs. 60.0%). Estimated GFRCKD-EPI was similar and all participants had residual urine output. Incident dialysis patients on PD were younger (57.1 ±12.1 vs. 65.1 ±12.0 years) and less often men (56.2% vs. 83.3%), and had lower BMI (23.5 ±3.0 vs. 26.6 ±3.8 kg/m2), higher diastolic blood pressure (87.3 ±16.0 vs. 77.4 ±9.2 mmHg), and less fluid overload (0.8 ±1.4 vs. 1.9 ±2.4 L), although not all differences were statistically significant. One PD patient switched to HD during follow-up.”12) In this table, in the 18% of patient with CKD have a history of Ktx, did they have an immunosuppressive drugs? This must be stated and clear in the characteristics of the patients. It is very different to study endothelial dysfunction to have or not IS drugs.We agree that this information is relevant. Therefore, we have added data on immunosuppressive medication use in participants with a history of KTx in both cross-sectional and longitudinal analyses, where available, in Table and S2 Table. In addition, we have added more detailed information to S1 Methods.S1 Methods:“For the 14 participants who were included in the cross-sectional analyses and had a history of kidney transplantation, data on their immunosuppressive regimen were retrospectively collected from the electronic patient health record. Of the 8 participants with CKD5-ND and a history of kidney transplantation, 7 participants were on immunosuppressive therapy of whom the therapeutic regimen could be retrieved for 6 participants: 1 prednisolone monotherapy, 4 TAC monotherapy, 1 either TAC or MMF monotherapy . Of the 3 participants with CKD5-HD and a history of kidney transplantation, 1 was on TAC monotherapy, 1 on MMF monotherapy and 1 did not use immunosuppressive therapy. Similarly, of the 3 participants with CKD5-PD and a history of kidney transplantation, 1 was one TAC monotherapy, 1 on MMF monotherapy and 1 did not use immunosuppressive therapy.All incident HD patients with a history of kidney transplantation were on immunosuppressive medication: 2 TAC monotherapy from baseline throughout follow-up, 1 either TAC or MMF monotherapy  from baseline up to and including the six month follow-up measurement, and for 1 of these participants the exact immunosuppressive regimen could not be retrieved.Similarly, all incident PD patients with a history of kidney transplantation were on immunosuppressive medication: 1 prednisolone from baseline throughout follow-up and 1 TAC monotherapy from baseline up to and including the six months follow-up measurement.All kidney transplant recipients with a history of kidney transplantation were on immunosuppressive therapy: 1 TAC monotherapy and 2 MMF monotherapy.”Further, we have performed sensitivity analyses excluding participants with prior KTx to explore confounding effects of immunosuppressive use and the presence of a KTx transplant. For cross-sectional analyses, results were similar. In addition, for longitudinal analyses, results were similar for incident HD and PD patients. Further, for kidney transplant recipients, results were largely similar, except for the decline in hs-CRP, SAA and IL-6, which was more pronounced.We have reported these results as follows in the additional analyses section of the Results :“Third, results of cross-sectional analyses were similar after exclusion of participants with a history of KTx (results not shown).Fourth, results of longitudinal analyses were similar for incident HD and PD patients after exclusion of participants with a history of KTx (results not shown). In addition, for kidney transplant recipients, results were largely similar, except for the decline in hs-CRP, SAA and IL-6 which was more pronounced after exclusion of participants with prior KTx (S8 Table).” 13) Important clinical data are missing: tobacco, alcool, lipids concentration, calcium phosphore bicarbonate potassium, hb etc. Medications used, mainly immunosuppressive drugs and corticoids, antihypertensive drugs, HMG CoA inhibitors, anticoagulants, aspirin etc. All this drugs could play an important role on endothelial function and inflammation.We agree with the reviewer that addition of these data would improve the manuscript. Therefore, we have added smoking status, use of renin-angiotensin-aldosterone system inhibitors and HMG CoA inhibitors to Table 1 and S1 Table. The use of immunosuppressive medication is already described in detail for kidney transplant recipients in S1 Methods. Unfortunately, data on the suggested laboratory values, which may provide explanatory information for the associations reported, as well as data on anticoagulant and aspirin use have not been collected for the present study. Methods : “Current smoking was based on self-report.”In addition, in line with the comment, we explored whether additional adjustment for systolic blood pressure, diastolic blood pressure, use of renin-angiotensin-aldosterone system inhibitors, use of HMG CoA inhibitors, current smoking and bmi would change results: results were similar.Results : “The above results were not materially changed after additional adjustment for systolic or diastolic blood pressure, use of renin-angiotensin-aldosterone system inhibitors, use of HMG-CoA inhibitors, current smoking or BMI (results not shown). “14) For the table 2, supplementary analysis separating HD and PD must be done. Adjustment with blood pressure could be of interest mainly regarding endothelial dysfunction.We now present the analyses in Table 2 stratified for CKD5-HD and CKD5-PD.Accordingly, the Results section  has been adapted as well:“Table S1 and Figure 1 shows the distributions of serum biomarkers of ED and LGI stratified by participant group.As compared with controls and after adjustment for age, sex and diabetes mellitus (Table 2), sVCAM-1, E-selectin, thrombomodulin, hs-CRP, SAA, IL-6, and TNF-α were higher in CKD5-ND, CKD5-HD and CKD5-PD. In addition, sICAM-1 was higher in CKD5-ND and CKD5-PD, and sICAM-3 was higher in CKD5-PD only.These results of individual serum biomarkers were also reflected by higher Z-scores for ED and LGI in CKD5-ND, CKD5-HD and CKD5-PD.As compared with CKD5-ND and after adjustment for age, sex and diabetes mellitus (Table 2), biomarkers of ED and LGI were in general similar in CKD5-HD. In addition, as compared with both CKD5-ND and CKD5-HD, sVCAM-1, E-selectin, thrombomodulin, sICAM-3 were higher in CKD5-PD. Further, as compared with CKD5-HD, sICAM-1 was higher in CKD5-PD. These results of individual serum biomarkers were also reflected by a higher Z-score for ED, but not LGI, in CKD5-PD as compared with CKD5-ND and CKD5-HD.”In addition, we have adjusted the first paragraph of the Discussion :“First, participants ESRD had higher levels of most serum biomarkers of ED and LGI than controls. Second, in participants with CKD5-HD, levels of serum biomarkers of ED and LGI were largely similar to those in CKD5-ND. In contrast, participants with CKD5-PD had higher levels of biomarkers of ED than participants with CKD5-ND and CKD5-HD.”Additional analyses with adjustment for systolic or diastolic blood pressure as well as renin-angiotensin-aldosterone system inhibitor use, HMG CoA use, current smoking and BMI, other import risk factors for ED, were similar. We have added this the Results section : “The above results were not materially changed after additional adjustment for systolic or diastolic blood pressure, use of renin-angiotensin-aldosterone system inhibitors, use of HMG-CoA inhibitors, current smoking or BMI (results not shown).”15) For the longitudinal analysis we must have separation of PD and HD patients. In addition, in my opinion for dialysis patient regarding the boxplot there is no real differences in all the biomarkers, the test used for statistical significance must be provide explicitly in the results. More interesting than a box blot the authors must provide individual courses of the biomarkers separating HD and PD.Longitudinal analyses stratified by dialysis modality were in the supplementary analyses. We have now moved these analyses to the main manuscript (Table 2). In addition, we have added individual trajectories to the box plots as requested, both for incident HD patients  and incident PD patients .Accordingly, the Results section  has been adapted as well: “Figure 2, Figure 3 and S3 Table show the distributions of serum biomarkers of ED and LGI in incident HD and incident PD patients from baseline up to six months after dialysis initiation.In linear mixed model analyses (Table 3), incident HD patients showed an increase in sVCAM-1, P-selectin and TNF-α from baseline to six months after dialysis initiation, although this change was only borderline statistically significantly for sVCAM-1. In addition, hs-CRP, SAA and IL-6 were lower after six months of follow-up.In linear mixed model analyses (Table 3), incident PD patients showed a statistically significant increase in sVCAM-1, thrombomodulin, sICAM-1 and sICAM-3 from baseline to six months after dialysis initiation. In these analyses, statistically significant interaction terms suggested that the courses of P-selectin, sICAM-1, sICAM-3, hs-CRP, SAA and IL-6 were different between incident HD and incident PD patients (Pinteraction < 0.10).”Please note that we have reanalyzed the data with the more modern linear mixed model technique. In contrast to repeated measures ANOVA, linear mixed model analysis provides an effect size  in addition to a test of statistical significance, which is presented in the Tables as well. Further, it allows selecting outliers based in regression diagnostics to assess the robustness of results (see below).This is addressed in the Methods section :“Longitudinal analyses were conducted stratified for incident HD and PD patients, and for kidney transplant recipients. Courses of natural log transformed serum biomarkers of ED and LGI over time following dialysis initiation and kidney transplantation were analyzed with linear mixed models to account for within-person correlations between repeated measurements and missing data. The fixed-effects part contained time as categorical variable (baseline served as reference), and the random-effects parts included a random intercept. The models for incident HD and PD patients also contained an interaction term between time and dialysis modality to test whether courses differ between HD and PD. Models were fitted by restricted maximum likelihood. Regression coefficients were exponentiated to obtain the ratio of (geometric mean) biomarker levels relative to baseline levels.”In addition, we have adjusted the first paragraph of the Discussion :“Third, incident PD patients showed an increase in several serum biomarkers of ED as well, but no convincing change in serum biomarkers of LGI. In contrast, results for incident HD patients were mixed, with an increase in some serum biomarkers of ED and LGI, but a decrease in other biomarkers of LGI.”16) We must have comparisons with other biological values, like calcium phosphore, bicarbonate urea, creatininemia etc.We agree that these data would improve the manuscript. Unfortunately, however, these data were not available for the present study, except for serum creatinine, which we have added to Table 1 and S2 Table .17) For transplantation patients same remarks regarding the representation of data. It could be of interest to indicate the course of values for the 4 patients undergoing dialysis then transplantation the data.Similar to the presentation of data of dialysis patients, we now provide boxplots with individual trajectories for kidney transplant recipients . In addition, we have reanalyzed the data with linear mixed model analyses as well.This has been adapted in the Results section :“In linear mixed model analyses (Table 4), kidney transplant recipients showed a statistically significant reduction in sVCAM-1, thrombomodulin, sICAM-3, and TNF-α at three months post-transplantation, which was still present after six months of follow-up. In addition, E-selectin, hs-CRP, SAA and IL-6 were lower at three and six months post-transplantation with similar or even larger magnitudes of effect , albeit not statistically significantly and with wide 95% confidence intervals.In addition, we have adjusted the first paragraph of the Discussion :“Fourth, following kidney transplantation levels of several serum biomarkers of ED and LGI were lower at three months post-transplantation and remained stable at three months thereafter.”Although we agree that a comparison of preemptively and non-preemptively transplanted patients would be interesting, the statistical power of the present study is too low to perform such an analyses and risk of chance findings is high. Instead, we have mentioned this as study limitation : “First, statistical power was limited, in particular for the comparison of HD and PD, and for the follow-up of kidney transplant recipients. In addition, the limited number of kidney transplant recipients precluded further stratification, for example to compare participants with preemptive and non-preemptive kidney transplantation.”18) The additional analysis section is a non sense. All this analysis must be included with main analysis or not discusses. The patients with cyst infection must be definitively excluded.The correlation plots  and stratified analyses for HD vs. PD have now been included in the main analyses . In addition, the explorative analyses on associations of eGFRCKD-EPI, eGFRresidual, residual urine output and dialysis vintage have been removed, as requested.We respectfully disagree with the reviewer that the patient with a history of recurring cyst infection should be excluded a priori. Indeed, his most recent episode was six months before study participation and a recent transluminal angioplasty of the patient’s dialysis shunt may be an alternative explanation for the increased inflammatory parameters [2]. Nevertheless, we agree that this was not clear from our formulation in the previous version of the manuscript. In addition, other participants may have had a rise in inflammatory markers due to an intercurrent infection or inflammatory response as well. Therefore, we have now more performed sensitivity analyses after exclusion of outliers . In general, results were largely similar after exclusion of outliers.Results : “First, when we excluded outliers  in the cross-sectional linear regression analyses, results were largely similar (S5 Table). Most notable dissimilarities after exclusion of outliers were a smaller difference between CKD5-HD and CKD5-PD for E-selectin; more pronounced differences in SAA between CKD5-HD/CKD5-PD and CKD5-ND; and more pronounced differences in Z-score for LGI between CKD5-PD and CKD5-ND/CKD5-HD.Second, when we excluded outliers in analyses for the courses of serum biomarkers following dialysis initiation (S6 Table) and kidney transplantation (S7 Table), results were in general similar or somewhat more pronounced.”In addition, we consider the additional adjustments for eGFRresidual, residual urine output, dialysis vintage meaningful: these analyses explore potential explanations for differences between CKD5-HD and CKD5-ND, but may distract from the main message if placed in the main results.Therefore, we have retained these results as part of the additional analyses .In addition, the use of linear mixed model analyses allowed exploring whether the course of eGFRresidual explained the courses of the serum biomarkers following HD or PD initiation. As we think this question naturally follows from the results, we have added it to the additional analyses : “Sixth, additional adjustment for eGFRresidual in linear mixed model analyses attenuated the courses of sVCAM-1 in incident HD and thrombomodulin in incident PD, but did not explain the courses in the other serum biomarkers (S9 Table).”19) The discussion will be analyzed after the modification asked for the methods and results.We have incorporated the results of the stratified analyses of HD and PD throughout the Discussion. Response to the Reviewer #2 In the present study, the authors compared biomarkers of endothelium dysfunction and low-grade inflammation in controls and in different groups of CKD patients. They show that most biomarkers are higher in CKD patients than in controls, higher in dialysis patients than in non-dialyzed patients and decline after kidney transplantation. Higher levels of serum biomarkers of ED and LGI in CKD patients have been previously shown in the literature. In addition, the data on serum biomarkers after kidney transplantation do not provide new informations and only confirm previous studies.We thank the reviewer for the time spend to review the manuscript.1) The design of the study is complex. It mixes the data of three separate observational studies, but the number of subjects per group is quite low. The characteristics of CKD5-ND and CKD5-D patients are different in terms of origin of end stage renal disease, and presence of diabetes mellitus.We agree with the reviewer that the design is complex. To help the reader in appreciating the flow of patients of the respective study groups, we have provided a flow diagram in S1 Fig. The limited study power is addressed as study limitation : “. First, statistical power was limited, in particular for the comparison of HD and PD, and for the follow-up of kidney transplant recipients. In addition, the limited number of kidney transplant recipients precluded further stratification, for example to compare participants with preemptive and non-preemptive kidney transplantation.”We agree that characteristics of CKD5-ND and CKD5-D patients differ in terms to cause of ESRD and prevalence of diabetes mellitus. Therefore, comparisons between CKD5-ND and CKD5-D were adjusted for diabetes mellitus. Unfortunately, the number of participants was too low to stratify by cause of ESRD. We added this to the study limitations : “Third, the distribution of ESRD causes, with varying prognosis, in this study may hamper comparisons between CKD5-ND and CKD5-D patients, and may limit generalizability. For example, the prevalence of polycystic kidney disease was high, whereas that of diabetic nephropathy was low.”2) Patients on PD are different from patients on HD , and additional analysis demonstrated differences in biomarkers in these two groups of patients. Therefore,the authors should not include these patients in the same CKD5D group.We have now provided results stratified by dialysis modality, both for the cross-sectional and longitudinal analyses. The Results section, including Tables and Figures, has been adapted accordingly.Results :“Population characteristics – cross-sectional analysesThirty-six controls, 43 participants with CKD5-ND, 20 participants with CKD5-HD and 14 participants with CKD5-PD comprised the cross-sectional study population . In general, participants with CKD5-ND, CKD5-HD and CKD5-PD were more often men and had a worse CVD risk profile than controls.Participants with CKD5-HD and CKD5-D more often had diabetes mellitus than those with CKD5-ND. All participants with CKD5-ND and most with CKD5-D had residual urine output. Participants with CKD5-PD slightly younger, less often men, less often had a history of CVD, and had higher residual GFR , slightly more fluid overload and shorter dialysis vintage than participants with CKD5-HD, although not all differences were statistically significant in this relatively small study population.” Results :“Associations of end-stage renal disease with serum biomarkers of endothelial dysfunction and low-grade inflammationTable S1 and Figure 1 shows the distributions of serum biomarkers of ED and LGI stratified by participant group.As compared with controls and after adjustment for age, sex and diabetes mellitus (Table 2), sVCAM-1, E-selectin, thrombomodulin, hs-CRP, SAA, IL-6, and TNF-α were higher in CKD5-ND, CKD5-HD and CKD5-PD. In addition, sICAM-1 was higher in CKD5-ND and CKD5-PD, and sICAM-3 was higher in CKD5-PD only.These results of individual serum biomarkers were also reflected by higher Z-scores for ED and LGI in CKD5-ND, CKD5-HD and CKD5-PD.As compared with CKD5-ND and after adjustment for age, sex and diabetes mellitus (Table 2), biomarkers of ED and LGI were in general similar in CKD5-HD. In addition, as compared with both CKD5-ND and CKD5-HD, sVCAM-1, E-selectin, thrombomodulin, sICAM-3 were higher in CKD5-PD. Further, as compared with CKD5-HD, sICAM-1 was higher in CKD5-PD. These results of individual serum biomarkers were also reflected by a higher Z-score for ED, but not LGI, in CKD5-PD as compared with CKD5-ND and CKD5-HD.”Results :“Population characteristics – longitudinal analysesThirty-four participants with CKD5-ND who initiated dialysis , 6 with CKD5-ND who received a kidney transplant, and 5 on chronic dialysis who received a kidney transplant were included in the longitudinal analyses . In addition, 4 participants who initiated dialysis and later received a kidney transplant were included as kidney transplant recipients as well. As compared with kidney transplant recipients, incident HD and PD patients were older (65.1 ±12.0 and 57.1 ±12.1 years vs. 51.6 ±12.8 years) and more often had a history of CVD (38.9% and 31.2% vs. 13.3%). In addition, incident HD patients were more often men (83.3% vs. 60.0%). Estimated GFRCKD-EPI was similar and all participants had residual urine output. Incident dialysis patients on PD were younger (57.1 ±12.1 vs. 65.1 ±12.0 years) and less often men (56.2% vs. 83.3%), and had lower BMI (23.5 ±3.0 vs. 26.6 ±3.8 kg/m2), higher diastolic blood pressure (87.3 ±16.0 vs. 77.4 ±9.2 mmHg), and less fluid overload (0.8 ±1.4 vs. 1.9 ±2.4 L), although not all differences were statistically significant. One PD patient switched to HD during follow-up.”Results :“Courses of serum biomarkers of endothelial dysfunction and low-grade inflammation following dialysis initiationFig 2, Fig 3 and S3 Table provide data on the distributions and individual trajectories of serum biomarkers of ED and LGI in incident HD and incident PD patients from baseline up to six months after dialysis initiation. In linear mixed model analyses (Table 3), incident HD patients showed an increase in sVCAM-1, P-selectin and TNF-α from baseline to six months after dialysis initiation, although this change was only borderline statistically significantly for sVCAM-1. In addition, hs-CRP, SAA and IL-6 were lower after six months of follow-up.In linear mixed model analyses (Table 3), incident PD patients showed a statistically significant increase in sVCAM-1, thrombomodulin, sICAM-1 and sICAM-3 from baseline to six months after dialysis initiation. In these analyses, statistically significant interaction terms suggested that the courses of P-selectin, sICAM-1, sICAM-3, hs-CRP, SAA and IL-6 were different between incident HD and incident PD patients (Pinteraction < 0.10).”In addition, we have adjusted the first paragraph of the Discussion :“This study on ED and LGI on individuals transitioning from untreated ESRD to renal replacement therapy had four main findings. First, participants ESRD had higher levels of most serum biomarkers of ED and LGI than controls. Second, in participants with CKD5-HD, levels of serum biomarkers of ED and LGI were largely similar to those in CKD5-ND. In contrast, participants with CKD5-PD had higher levels of biomarkers of ED than participants with CKD5-ND and CKD5-HD. Third, incident PD patients showed an increase in several serum biomarkers of ED as well, but no convincing change in serum biomarkers of LGI. In contrast, results for incident HD patients were mixed, with an increase in some serum biomarkers of ED and LGI, but a decrease in other biomarkers of LGI. Fourth, following kidney transplantation levels of several serum biomarkers of ED and LGI were lower at three months post-transplantation and remained stable at three months thereafter.”Further, we have incorporated the results of analyses stratified by dialysis modality throughout the Discussion.3) The choice of statistical analyses is not clear. Why do the authors compare the data of biomarkers between groups with linear regression analysis and not with tests for group comparison like ANOVA or Mann Whitney? In addition, why do the authors use a natural log transformation to perform statistical test on the levels of serum biomarkers? How is the distribution of the data ?We chose to analyze between-group differences with linear regression analyses as this technique allows adjustment for potential confounders .The distribution of serum biomarkers were positively skewed. Therefore, these values were natural log transformed to obtained acceptably normally distributed residuals of the regression analyses.We have now clarified this in the Methods : “Differences in individual serum biomarkers of ED and LGI between CKD5-ND, CKD5-HD, CKD5-PD and controls were evaluated with linear regression analyses to allow adjustment for potential confounders. Levels of serum biomarkers were positively skewed and, therefore, natural log transformed to obtain acceptably normally distributed residuals. The regression coefficients were exponentiated to obtain the ratio of (geometric mean) levels of the serum biomarkers in CKD5-ND, CKD5-HD and CKD5-PD relative to controls and among ESRD groups.”Please note that we have reanalyzed the data with the more modern linear mixed model technique, which in contrast to repeated measures ANOVA, provides an effect size  in addition to a test of statistical significance, which is presented in the Tables as well.This is addressed in the Methods section :“Longitudinal analyses were conducted stratified for incident HD and PD patients, and for kidney transplant recipients. Courses of natural log transformed serum biomarkers of ED and LGI over time following dialysis initiation and kidney transplantation were analyzed with linear mixed models to account for within-person correlations between repeated measurements and missing data. The fixed-effects part contained time as categorical variable (baseline served as reference), and the random-effects parts included a random intercept. The models for incident HD and PD patients also contained an interaction term between time and dialysis modality to test whether courses differ between HD and PD. Models were fitted by restricted maximum likelihood. Regression coefficients were exponentiated to obtain the ratio of (geometric mean) biomarker levels relative to baseline levels.”4) Identical data are presented several times, in different figures/tables. For example, Figure 2 and Table 3 provides the same informations, data in Table 3 are also in S1 Table etc…We have adapted the tables to reduce overlapping information in the main manuscript. However, we think that some readers prefer tabulated data on serum biomarkers over boxplots. Therefore, we have provided such tables as supplementary data .5) For a better understanding, the authors should add the symbols of p values on figures and tables. In particular, the p values between CKD5-ND and CKD5D should be mentioned in Table 1.We have added symbols for P values for comparisons among patient groups to Table 1 as requested and added this to the Methods section as well.Methods :“Unadjusted post-hoc comparisons of characteristics of participant groups were performed with the independent Student t-test for normally distributed data, Dunn’s test for non-normally distributed data and Fisher’s exact test for categorical data if a one-way analysis of variance (ANOVA) test, Kruskal-Wallis test and Fisher’s exact test, respectively, indicated an overall difference between groups.”Legend Table 1 : “† P value < 0.05 vs. controls; ‡ P value < 0.05 vs. CKD5-ND; ¶ P value < 0.05 vs. CKD5-HD. Unadjusted post-hoc comparisons of controls, CKD5-ND, CKD5-HD and CKD5-PD were performed with the independent Student t-test for normally distributed data, Dunn’s test for non-normally distributed data and Fisher’s exact test for categorical data if an ANOVA, Kruskal-Wallis test and Fisher’s exact test, respectively, indicated an overall difference between groups .”Legend S2 Table: “¶ P value < 0.05 vs. CKD5-HD based on Student’s t test for normally distributed data, Wilcoxon rank sum test for non-normally distributed data and Fisher’s exact test for categorical data.”In addition, we have added figures for P values to Fig 1-4, as requested.6) In table 3, an entire column is NA, please remove the column. In S1 Table, dialysis vintage and KT/V are NA, please remove the lines.We have adapted the Tables as requested. Please note that former Table 3 is now S3 Table and S4 Table, and that former S1 Table is now S2 Table. Response to Reviewer #3Manuscript is well written. Only minor suggestions to improve the manuscript.We thank the reviewer for the compliment and the time spend to review the manuscript.1) Please try to avoid the use of  in the descriptive sentences in academic writing “This risk is not (completely) mitigated by renal replacement therapy”We have removed the brackets as requested. Abstract : “This risk is not (completely) mitigated by renal replacement therapy.”Methods : “History of diabetes mellitus and cardiovascular disease, and medication use were based on self-report for healthy controls and the health record for patients.”2) “Only data of their first study was analyzed” “of” should be “from”We have changed this accordingly : “Only data from their first study was analyzed.”3) Also, I suggest the investigators to may improve the introduction/discussion by going over very nice review on this topic PMID: 30607032 Nat Rev Nephrol. 2019 Feb;15(2):87-108. doi: 10.1038/s41581-018-0098-zWe thank the reviewer for the recommended review. As the review is mostly on the effects of ED at the renal level instead of systemic ED, we briefly mention this in the Introduction : “In addition, ED and LGI interact at the renal level and may contribute to the development and progression of renal injury, as elegantly described recently[3].”In addition, we added this review as a reference to our discussion on the causal mechanisms of ED and LGI in ESRD : “The causal mechanisms of ED and LGI in ESRD are likely multifactorial and may include exposure to traditional CVD risk factors, oxidative stress, fluid and sodium overload, gut dysbiosis, accumulation of uremic toxins[3-5], and turbulent flow[6] due to the hyperdynamic circulation after arteriovenous shunt creation[7]. In addition, ED and LGI may be interrelated[8] as illustrated by the correlation matrix.” Journal Requirements:1. When submitting your revision, we need you to address these additional requirements.Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttp://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfWe have adapted the manuscript according to PLOS ONE’s style requirements. The heading of the section listing supporting information was changed to Supporting information . 2. In your Methods section, please provide additional information regarding the source of donated organs received by the patients in the study. Please specify whether the study involved the use of donated tissue/organs from any vulnerable populations, and provide information on the consent given by the donor or their next of kin. Examples of vulnerable populations include prisoners, subjects with reduced mental capacity due to illness or age, and children. If such a population was used, please ensure you have describe the population and justify the decision to use tissue/organ donations from this group. If not, please state in your Ethics Statement, 'None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given.We have added this to the Methods section : In addition, none of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given.Donors are carefully provided information and the screening procedure of a potential donor is essential in living kidney donation. Therefore, potential donors are screened and evaluated according to the national Guideline ‘Evaluation of potential donors for living kidney donation’ of the Dutch Transplant Society and the Dutch Kidney Transplantation Advisory Committee. These guidelines are based on the ‘The United Kingdom Guidelines for Living Donor Kidney Transplantation’ of the British Transplantation Society and the Renal Association (see link) https://bts.org.uk/wp-content/uploads/2018/01/BTS_LDKT_UK_Guidelines_2018.pdf. All donors have been informed about the risks and consequences of kidney donation, and before donation written informed consent is needed. Independent psychosocial evaluation is mandatory to be sure that the potential donor is well informed and understands the risks of donation and a further life with a reduced eGFR.3. 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Balloon angioplasty and stent implantation induce a vascular inflammatory reaction. J Endovasc Ther. 2002;9(1):59-66. Epub 2002/04/18. doi: 10.1177/152660280200900111.3. Jourde-Chiche N, Fakhouri F, Dou L, Bellien J, Burtey S, Frimat M, et al. Endothelium structure and function in kidney health and disease. Nature reviews Nephrology. 2019;15(2):87-108. Epub 2019/01/05. doi: 10.1038/s41581-018-0098-z.4. Cobo G, Lindholm B, Stenvinkel P. Chronic inflammation in end-stage renal disease and dialysis. Nephrol Dial Transplant. 2018;33(suppl_3):iii35-iii40. Epub 2018/10/04. doi: 10.1093/ndt/gfy175.5. Malyszko J. Mechanism of endothelial dysfunction in chronic kidney disease. Clin Chim Acta. 2010;411(19-20):1412-20. Epub 2010/07/06. doi: 10.1016/j.cca.2010.06.019.6. Davies PF. Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med. 2009;6(1):16-26. Epub 2008/11/26. doi: 10.1038/ncpcardio1397.7. Meeus F, Kourilsky O, Guerin AP, Gaudry C, Marchais SJ, London GM. Pathophysiology of cardiovascular disease in hemodialysis patients. Kidney Int Suppl. 2000;76:S140-7. Epub 2000/08/11.8. Padilla J, Simmons GH, Bender SB, Arce-Esquivel AA, Whyte JJ, Laughlin MH. Vascular effects of exercise: endothelial adaptations beyond active muscle beds. Physiology (Bethesda). 2011;26(3):132-45. Epub 2011/06/15. doi: 10.1152/physiol.00052.2010.AttachmentRespons to Reviewers.docxSubmitted filename: Click here for additional data file. 3 Sep 2019[EXSCINDED]Endothelial dysfunction and low-grade inflammation in the transition to renal replacement therapyPONE-D-19-18004R1Dear Dr. van Gennip,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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